Stem cells are undifferentiated cells that exist in many tissues of embryos and adult mammals. Both adult and embryonic stem cells are able to differentiate into a variety of cell types and, accordingly, may be a source of replacement cells and tissues that are damaged in the course of disease, infection, or because of congenital abnormalities. (See, e.g., Lovell-Badge (2001) Nature 414:88-91; Donovan et al. (2001) Nature 414:92-97). Various types of putative stem cells exist which; when they differentiate into mature cells, carry out the unique functions of particular tissues, such as the heart, the liver, or the brain. Pluripotent stem cells are thought to have the potential to differentiate into almost any cell type, while multipotent stem cells are believed to have the potential to differentiate into many cell types (Robertson (1997) Meth. Cell Biol. 75:173; and Pedersen (1994) Reprod. Fertil. Dev. 6:543). For example, human induced pluripotent stem cells (hiPSCs) are pluripotent cells derived from somatic cells by the ectopic expression of reprogramming factors (see for example Nakagawa et al, (2008) Nat. Biotechnol 26:101-106). These cells share all the key characteristics of human embryonic stem cells (hESC) and can be generated from cells isolated from human patients with specific diseases (see for example Dimos et al, (2008) Science 321:1218-1221).
Stable transgenesis and targeted gene insertion into stem cells have a variety of applications. Stably transfected stem cells can be used as a cellular vehicle for protein-supplement gene therapy and/or to direct the stem cells into particular lineages. See, e.g., Eliopoulos et al. (2008) Blood Cells, Molecules, and Diseases 40(2):263-264. In addition, insertion of lineage-specific reporter constructs would allow isolation of lineage-specific cells, and would allow drug discovery, target validation, and/or stem cell based studies of gene function and the like based upon those results. For example, U.S. Pat. No. 5,639,618 describes in vitro isolation of a lineage-specific stem cell by transfecting a pluripotent embryonic stem cell with a construct comprising a regulatory region of a lineage-specific gene operably linked to a DNA. However, current strategies of stem cell transfection often randomly insert the sequence of interest (reporter) into the stem cell. See, e.g., Islan et al. (2008) Hum Gene Ther October; 19(10):1000-1008; DePalma et al. (2005) Blood 105(6):2307-2315. The inability to control the location of genome insertion can lead to highly variable levels of expression throughout the stem cell population due to position effects within the genome.
Additionally, current methods of stable transgenesis and amplification of transgenes often result in physical loss of the transgene, transgene silencing over time or upon stem cell differentiation, insertional mutagenesis by the integration of a transgene and autonomous promoter inside or adjacent to an endogenous gene, the aberrant expression of endogenous genes caused by the heterologous regulatory elements associated with the randomly integrating transgene, the creation of chromosomal abnormalities and expression of rearranged gene products (comprised of endogenous genes, the inserted transgene, or both), and/or the creation of vector-related toxicities or immunogenicity in vivo from vector-derived genes that are expressed permanently due to the need for long-term persistence of the vector to provide stable transgene expression. Furthermore, the correct expression pattern of a given endogenous gene such as a gene that is a lineage marker emerges out of the combined action of a large number of cis-regulatory elements. See, e.g. Levasseur et al (2008) Genes Dev. 22: 575-580.
Zinc finger nucleases can be used to efficiently drive targeted gene insertion at extremely high efficiencies using a homologous donor template to insert novel gene sequences via homology-driven repair (HDR). See, for example, United States Patent Publications 20030232410; 20050208489; 20050026157; 20050064474; and 20060188987, and International Publication WO 2007/014275, the disclosures of which are incorporated by reference in their entireties for all purposes. Zinc finger nuclease-driven gene insertion can use the transient delivery of a non-integrating vector, and this does not require long-term persistence of the delivery vector, avoiding issues of insertional mutagenesis and toxicities or immunogenicity from vector-derived genes.
However, there remains a need for controlled, site-specific integration into a stem cell population.